Genome-Wide Identification and Analysis of MKK and MAPK Gene

International Journal of Molecular Sciences

Article Genome-Wide Identiﬁcation and Analysis of MKK and MAPK Gene Families in Brassica Species and Response to Stress in Brassica napus

Zhen Wang 1,2,3,† , Yuanyuan Wan 1,2,3,†, Xiaojing Meng 1,2,3, Xiaoli Zhang 1,2,3, Mengnan Yao 1,2,3, Wenjie Miu 1,2,3, Dongming Zhu 1,2,3, Dashuang Yuan 1,2,3, Kun Lu 1,2,3 , Jiana Li 1,2,3, Cunmin Qu 1,2,3,* and Ying Liang 1,2,3,*

1 College of Agronomy and Biotechnology, Southwest University, Chongqing 400715, China; [email protected] (Z.W.); [email protected] (Y.W.); [email protected] (X.M.); [email protected] (X.Z.); [email protected] (M.Y.); [email protected] (W.M.); [email protected] (D.Z.); [email protected] (D.Y.); [email protected] (K.L.); [email protected] (J.L.) 2 Academy of Agricultural Sciences, Southwest University, Chongqing 400715, China 3 Chongqing Engineering Research Center for Rapeseed, Chongqing 400715, China * Correspondence: [email protected] (C.Q.); [email protected] (Y.L.); Tel.: +86-23-6825-0701 (C.Q. & Y.L.) † These authors contributed equally to this work.

Abstract: Mitogen-activated protein kinase (MAPK) cascades are common and conserved signal transduction pathways and play important roles in various biotic and abiotic stress responses and growth and developmental processes in plants. With the advancement of sequencing technology,

more systematic genetic information is being explored. The work presented here focuses on two protein families in Brassica species: MAPK kinases (MKKs) and their phosphorylation substrates

Citation: Wang, Z.; Wan, Y.; Meng, MAPKs. Forty-seven MKKs and ninety-two MAPKs were identiﬁed and extensively analyzed from X.; Zhang, X.; Yao, M.; Miu, W.; Zhu, two tetraploid (B. juncea and B. napus) and three diploid (B. nigra, B. oleracea, and B. rapa) Brassica D.; Yuan, D.; Lu, K.; Li, J.; et al. species. Phylogenetic relationships clearly distinguished both MKK and MAPK families into four Genome-Wide Identiﬁcation and groups, labeled A–D, which were also supported by gene structure and conserved protein motif Analysis of MKK and MAPK Gene analysis. Furthermore, their spatial and temporal expression patterns and response to stresses Families in Brassica Species and (cold, drought, heat, and shading) were analyzed, indicating that BnaMKK and BnaMAPK transcript Response to Stress in Brassica napus. levels were generally modulated by growth, development, and stress signals. In addition, several Int. J. Mol. Sci. 2021, 22, 544. https:// protein interaction pairs between BnaMKKs and C group BnaMAPKs were detected by yeast two- doi.org/10.3390/ijms22020544 hybrid assays, in which BnaMKK3 and BnaMKK9 showed strong interactions with BnaMAPK1/2/7, suggesting that interaction between BnaMKKs and C group BnaMAPKs play key roles in the crosstalk Received: 9 December 2020 between growth and development processes and abiotic stresses. Taken together, our data provide Accepted: 5 January 2021 a deeper foundation for the evolutionary and functional characterization of MKK and MAPK gene Published: 7 January 2021 families in Brassica species, paving the way for unraveling the biological roles of these important

Publisher’s Note: MDPI stays neu- signaling molecules in plants. tral with regard to jurisdictional clai- ms in published maps and institutio- Keywords: MKKs; MAPKs; growth and development; abiotic stresses; interaction; Brassica napus nal afﬁliations.

1. Introduction Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. Plants have evolved specialized mechanisms that sense adverse environmental con- This article is an open access article ditions and adapt their physiology, growth, and developmental processes accordingly distributed under the terms and con- through the transcriptional, translational, and post-translational regulation of their en- ditions of the Creative Commons At- zymatic activities. Protein phosphorylation is the most common post-translational mod- tribution (CC BY) license (https:// iﬁcation observed across organisms and typically involves protein kinases in signaling creativecommons.org/licenses/by/ cascades [1]. Mitogen-activated protein kinase (MAPK) cascades are common signal trans- 4.0/). duction pathways in all eukaryotes [2] and play critical roles in signal transduction [3–5].

Int. J. Mol. Sci. 2021, 22, 544. https://doi.org/10.3390/ijms22020544 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 544 2 of 21

In plants, MAPKs transduce extracellular stimuli to induce a range of cellular responses, specifically post-translationally modifying proteins to modulate cellular functions associated with various physiological, developmental, and phytohormonal responses [4,6–8]. MAPK signaling cascades also result in the activation of cytoskeletal proteins, phospholi- pases, and microtubule-associated proteins, and can target transcription factors to modulate the expression of specific sets of genes in response to environmental stimuli [9–11]. A canonical MAPK signaling network is composed of three specific protein kinases, namely MAPK kinase kinases (MAPKKKs), MAPK kinases (MKKs), and MAPKs, which are activated sequentially by phosphorylation at conserved activation sites [1,7]. MAPKKKs are the first component in these phosphorelay cascades and phosphorylate and activate MKKs on S/T-X3–5-S/T motifs. All MKKs also have a putative MAPK docking domain (D domain) K/R-K/R-K/R-X1–6-L-X-L-/V/I in their N termini. MKKs are dual-specificity kinases that activate their downstream MAPKs through phosphorylation of the T-X-Y motif in the activation loop (T-loop) [4]. Activated MAPKs then lead to the phosphorylation of various downstream substrates, including transcription factors and other signaling components that regulate the expression of downstream genes [1]. A total of 10 MKKs and 20 MAPKs have been characterized in Arabidopsis thaliana, which revealed that Arabidopsis MKKs may activate different MAPKs and hence participate in signal integration from distinct signaling pathways [7,12]. MKKs can be classified into four major groups (A–D) based on the sequence of their S/T-X3–5-S/T motif and D domain. Similarly, MAPKs also form four groups (A–D) based on their conserved T-X-Y motif, with groups A–C harboring the T-E-Y sequence and group D the T-D-Y sequence [7]. Extensive studies in plants have revealed that MAPK cascades are indispensable for the regulation of the cell cycle, growth, and development, and responses to biotic and abiotic stresses, such as pathogen infection, salt, drought, heat, low temperature, reactive oxy- gen species (ROS), and heavy metals [8,13–17]. To date, several MAPK signaling pathways have been characterized in detail. The Arabidopsis MAPKKK1-MKK4/5-MAPK3/6 module was the first identified MAPK signaling module in plants, mediating the transcriptional upregulation of genes encoding the transcription factors WRKY22 and WRKY29 in response to attacks by both fungal and bacterial pathogens [18,19]. Additional modules have since been linked to the regulation of multiple and varied biological processes. For instance, the MKK4/5-MAPK3/6 module has been shown to play important roles in responses to biotic stress and during stomatal development [18,20,21]. The MKK1-MAPK3/6 and MKK6- MAPK4 branches are also key players in stomatal patterning and dynamics [13,20–22]. The MKK3-MAPK8 pathway mediates Ca2+ and ROS signaling during early wounding [23]. The MAPKKK1-MKK1/2-MAPK4 branch positively regulates defense responses against necrotrophic fungi, while negatively regulating defenses against biotrophic pathogens; this branch is also activated by drought, freezing, and salt stresses [24–27]. The MKK9- MAPK3/6 pathway was reported to regulate ethylene signaling and camalexin biosynthesis and might also play a role during leaf senescence [28–30]. The MKK6-MAPK4/11 pathway was shown to directly regulate cytokinesis and mitosis [31–34]. Another example is the abscisic acid (ABA)-activated MAPKKK17/18-MKK3-MAPK1/2/7/14 pathway, which transduces the ABA signal to regulate the expression of a number of ABA-dependent genes [35]. In previous work, we reported that overexpression of the rapeseed (Brassica napus) BnaMAPK1 gene increased plant resistance to drought stress and to the pathogenic fungus Sclerotinia sclerotiorum [36,37]. In contrast to A and B group MAPKs, reports on C groups are scarce. Fortunately, the sequence availability of multiple Brassica genomes is now allowing a thorough investigation of MKKs and MAPKs and their possible interactions. Here, we identified and classified MKKs and MAPKs in five Brassica species and investigated the expression patterns of BnaMKK and BnaMAPK in spatial and temporal expression patterns and in response to abiotic stresses. Furthermore, Y2H assays were performed on the potential interaction relationship between the encoded BnaMKKs and C group BnaMAPKs, revealing an important regulation of BnaMKK-BnaMAPK signaling modules in the complex crosstalk between growth and developmental processes and Int. J. Mol. Sci. 2021, 22, 544 3 of 21

abiotic stress responses. This work thus suggests a molecular strategy to coordinate growth and development and stress responses in B. napus and lay a solid foundation for further deciphering MAPK cascades and for future improvement of various defense responses using these signaling pathways for B. napus breeding.

2. Results 2.1. Identification of 47 MKK and 92 MAPK Genes in Five Brassica Species A systematic Basic Local Alignment Search Tool for Protein (BLASTP) search was performed using the Arabidopsis MKK and MAPK protein sequences as query against the genome databases for B. juncea (Bju), B. napus (Bna), B. nigra (Bni), B. oleracea (Bol), and B. rapa (Bra). 47 MKK genes were identified: 11 genes in B. juncea, 14 in B. napus, seven in B. nigra, six in B. oleracea, and nine in B. rapa (Table1). Similarly, 92 MAPK genes were identified, with 25 genes in B. juncea, 29 in B. napus, 18 in B. nigra, six in B. oleracea, and 14 in B. rapa (Table1). Not all Arabidopsis MKK and MAPK genes had clear orthologs in the other species: Orthologs for AtMKK2/9 or AtMAPK18/19 in B. juncea; AtMKK7 or AtMAPK3/5/11/12 in B. napus; AtMMK7/8/9 or AtMAPK4/8/14/17/18 in B. nigra; AtMKK3/5/7/10 or AtMAPK1/2/3/5/7- 16/20 in B. oleracea; and AtMKK2/7 or AtMAPK6/7/11/14/16/18/20 in B. rapa were not found. These Brassica MKK and MAPK genes were named according to their closest homologs in Arabidopsis, and multiple copies of one gene were named with 1, 2, or 3 (details in Table S1). This analysis indicated that the duplication and retention of MKK and MAPK genes were more frequent in tetraploids than in diploids.

Table 1. Number of Brassica genes orthologous to Arabidopsis (At) MKK and MAPK genes identiﬁed from genome databases for ﬁve Brassica species.

AtMAPK Signature Motif Group Bju Bna Bni Bol Bra AtMKK1 VGT(YP)YMSPER A 1 2 1 1 1 AtMKK2 VGT(YN)YMSPER A - 2 1 1 - AtMKK3 VGT(VT)YMSPER B 2 2 1 - 1 AtMKK4 VGT(IA)YMSPER C 1 1 1 1 1 MAPK AtMKK5 VGT(IA)YMSPER C 2 1 1 - 2 kinases AtMKK6 VGT(YN)YMSPER A 1 1 1 1 1 (MKK) AtMKK7 VGT(CA)YMSPER D 2 - - - - family AtMKK8 VGT(FA)YMSPER D 1 2 - 1 1 AtMKK9 VGT(CA)YMSPER D - 2 - 1 1 AtMKK10 VGT(CA)YMSPER D 1 1 1 - 1 Total 11 14 7 6 9 AtMAPK1 T(E)Y C 2 1 1 - 1 AtMAPK2 T(E)Y C 1 1 1 - 1 AtMAPK3 T(E)Y A 2 - 1 - 1 AtMAPK4 T(E)Y B 1 2 - 1 1 AtMAPK5 T(E)Y B 2 - 1 - 1 AtMAPK6 T(E)Y A 2 1 1 1 - AtMAPK7 T(E)Y C 1 2 2 - - AtMAPK8 T(D)Y D 1 2 - - 2 Mitogen- AtMAPK9 T(D)Y D 1 2 1 - 1 activated AtMAPK10 T(E)Y A 1 2 1 - 1 protein AtMAPK11 T(E)Y B 1 - 1 - - kinase AtMAPK12 T(E)Y B 2 - 2 - 1 (MAPK) AtMAPK13 T(E)Y B 2 2 1 - 1 family AtMAPK14 T(E)Y C 1 1 - - - AtMAPK15 T(D)Y D 1 2 1 - 1 AtMAPK16 T(D)Y D 2 1 1 - - AtMAPK17 T(D)Y D 1 2 - 1 1 AtMAPK18 T(D)Y D - 3 - 2 - AtMAPK19 T(D)Y D - 3 1 1 1 AtMAPK20 T(D)Y D 1 2 2 - - Total 25 29 18 6 14 Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 4 of 22

AtMAPK15 T(D)Y D 1 2 1 - 1 AtMAPK16 T(D)Y D 2 1 1 - - AtMAPK17 T(D)Y D 1 2 - 1 1 AtMAPK18 T(D)Y D - 3 - 2 - AtMAPK19 T(D)Y D - 3 1 1 1 Int. J. Mol. Sci. 2021, 22, 544 AtMAPK20 T(D)Y D 1 2 2 - 4 of- 21 Total 25 29 18 6 14

2.2. Phylogenetic Analysis of MKKs and MAPKs from Arabidopsis and Five BrassicaBrassica SpeciesSpecies To clarifyclarify thethe evolutionaryevolutionary relationshipsrelationships of MKKsMKKs andand MAPKsMAPKs inin ArabidopsisArabidopsis andand other Brassica species,species, multiple multiple protein protein alignments alignments of of the the full full-length-length MKK MKK and and MAPK MAPK se- sequencesquences were were performed performed to generate to generate unrooted unrooted phylogenetic phylogenetic trees trees with with the Neighbor the Neighbor--join- joininging (NJ) (NJ) method. method. Both Both multiple multiple alignments alignments and andthe phylogenetic the phylogenetic analysis analysis suggested suggested that thatMKK MKK proteins proteins were were classified classified into into four four groups: groups: MKK1/2/6 MKK1/2/6 (A (A group), group), MKK3 MKK3 (B (B group), group), MKK4/5MKK4/5 (C (C group), group), and and MKK7/8/9/10 MKK7/8/9/10 (D group) (D group) (Table (Table 1). Likewise,1). Likewise, MAPK MAPK proteins proteins were werealso classified also classified into fourinto four groups: groups: MAPK3/6/10 MAPK3/6/10 (A group), (A group), MAPK4/5/11/12/13 MAPK4/5/11/12/13 (B group), (B group),MAPK1/2/7/14 MAPK1/2/7/14 (C group), (C and group), MAPK8/9/15/16/17/18/19/20 and MAPK8/9/15/16/17/18/19/20 (D group) (Table (D group) 1, Figure (Table 1).1, FigureNotably1 )., BraMKK5.1 Notably, BraMKK5.1 clustered clusteredmore closely more with closely AtMKK4 with AtMKK4and BjuMKK7.2 and BjuMKK7.2 with AtMKK9 with AtMKK9(Figure 1a). (Figure Furthermore,1a). Furthermore, the four the MAPK10 four MAPK10 proteins proteins BnaMAPK10, BnaMAPK10, BraMAPK10, BraMAPK10, Bju- BjuMAPK10,MAPK10, andand BniMAPK10 BniMAPK10 grouped grouped more more closely closely with with AtMAPK6, AtMAPK6, while while BniMAPK11 BniMAPK11 and andBjuMAPK11 BjuMAPK11 were were more more closely closely related related to toAtMAPK14 AtMAPK14 in in the the phylogenetic phylogenetic tree tree (Figure(Figure 11b).b) . These results suggest that these proteins in thethe fivefive BrassicaBrassica speciesspecies andand ArabidopsisArabidopsis areare closely related, but also implyimply thatthat functionalfunctional differentiationdifferentiation maymay havehave occurredoccurred withinwithin multiple groups.

FigureFigure 1.1. Phylogenetic analysis of MKKs and MAPKs family members from Arabidopsis thaliana (At), Brassica juncea (Bju), BrassicaBrassica napus ((Bna),), BrassicaBrassica nigra nigra (Bni(Bni),), BrassicaBrassica oleracea oleracea (Bol(Bol), and), and BrassicaBrassica rapa rapa (Bra(Bra). (a).) Phylogenetic (a) Phylogenetic trees trees of MKKs of MKKs from fromArabidopsisArabidopsis and fiveand Brassica ﬁve Brassica species.species. (b) Phylogenetic (b) Phylogenetic trees trees of MAPKs of MAPKs from from ArabidopsisArabidopsis and andfive ﬁveBrassicaBrassica species.species. The Theun- unrootedrooted phylogenetic phylogenetic trees trees were were constructed constructed using using MEGA MEGA-X-X with with the the NJ NJ method; method; the the bootstrap bootstrap test was performed withwith 1000 iterations. Different colored backgrounds indicate different groups. 1000 iterations. Different colored backgrounds indicate different groups.

We nextnext focusedfocused onon MKKMKK andand MAPKMAPK genesgenes fromfrom B.B. napusnapus.. TheThe lengthlength ofof MKK coding sequences in B. napus covered a wide range, from 735 bp toto 15571557 bp,bp, withwith aa meanmean lengthlength of 1072.5 bp. The associated molecular weight (MW) across BnaMKK proteins varied from 27.1 kDa to 57.5 kDa, with a mean of 39.5 kDa; the isoelectric point (pI) ranged from 5.4 to 9.4, with a mean of 6.7 (Table S2). Similarly, the length of BnaMAPK coding sequences varied from 1110 bp to 1977 bp, with a mean length of 1505.3 bp. The mean MW of BnaMAPKs proteins was 56.9 kDa (range 42.3 kDa to 74.3 kDa), while pI values ranged from 4.8 to 9.3, with a mean of 7.1 (Table S2). Int. J. Mol. Sci. Sci. 2021,, 22,, x 544 FOR PEER REVIEW 5 of 22 21

of2.3. 1072.5 Gene bp. Structure The associated and Conserved molecular Motif weight Analysis (MW) of BnaMKKs across BnaMKK and BnaMAPKs proteins in varied B. napus from 27.1 kDaThe to genomic 57.5 kDa, position with a of mean all BnaMKK of 39.5 kDa;and BnaMAPKthe isoelectricgenes point were (pI) determined ranged from to better5.4 to 9.4,understand with a mean their chromosomalof 6.7 (Table S2). distribution Similarly, in theB. napuslength, anof allopolyploidBnaMAPK coding derived sequences from a variedcross between from 1110 the bp diploid to 1977 species bp, withB. rapa a mean(A subgenome length of progenitor)1505.3 bp. andThe B. mean oleraceae MW (C of BnaMAPKssubgenome proteins progenitor). was Nine56.9 kDaBnaMKK (rangegenes 42.3 mappedkDa to 74.3 to thekDa), A subgenomewhile pI values (Figure ranged2a); fromthe remaining 4.8 to 9.3, ﬁvewithBnaMKK a mean ofgenes 7.1 (Table mapped S2).to the C subgenome (Figure2b). In the case of the BnaMAPK gene family, 16 BnaMAPKs mapped to the A subgenome (Figure3a) and 2.3.13 BnaMAPK Gene Structuregenes and mapped Conserved to the Motif C subgenome,Analysis of BnaMKKs of which andBnaMAPK9.1 BnaMAPKs, BnaMAPK18.2 in B. napus , and BnaMAPK20.2The genomic positionwere located of all to BnaMKK the Cnn_random and BnaMAPK chromosome, genes were a collection determined of unmapped to better understandscaffolds tentatively their chromosomal assigned to distribution the C subgenome in B. napus (Figure, an3b). allopolyploid derived from a crossNext, between the the structures diploid ofspeciesBnaMKK B. rapaand (ABnaMAPK subgenomegenes progenitor) were examined. and B. oleraceaeBnaMKK (C subgenomegenes had between progenitor) one. and Nine nine BnaMKK exons, withgenes seven mappedBnaMKK to thegenes A subgenome (BnaMKK4 (Figure, BnaMKK5 2a);, theBnaMKK8.1 remaining, BnaMKK8.2 five BnaMKK, BnaMKK9.1 genes mapped, BnaMKK9.2 to the C ,subgenome and BnaMKK10 (Figure) consisting 2b). In the of case a sin- of thegle BnaMAPK and very longgene exon,family, much 16 BnaMAPKs longer than mapped any other to the exon A subgenome in the other (FigureBnaMKK 3a) angenesd 13 BnaMAPK(Figure4a) genes. The mapped number to of the exons C subgenome, among BnaMAPK of whichgenes BnaMAPK9.1 varied from, BnaMAPK18.2 two to 13,, with and BnaMAPK20.2three C group memberswere located (BnaMAPK1 to the Cnn_random, BnaMAPK2, chromosome, and BnaMAPK14 a collection) having only of unmapped two exons scaffolds(Figure4b). tentatively assigned to the C subgenome (Figure 3b).

Figure 2. ChromosomalChromosomal locat locationsions of of BnaMKKBnaMKK genesgenes in in B.B. napus napus genome.genome. (a)( aGenomic) Genomic position position of of BnaMKK genes in subgenome A. ( b) Genomic position of BnaMKK genes in subgenome C.

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Figure 3. Chromosomal locations of BnaMAPK genes in B. napus genome.genome. ( a) Genomic position of BnaMAPK genes in subgenome A. (b) Genomic position of BnaMAPK genes in subgenome C. subgenome A. (b) Genomic position of BnaMAPK genes in subgenome C.

Next,Then, the the structures conserved of motifs BnaMKK of BnaMKK and BnaMAPK and BnaMAPK genes were proteins examined. were BnaMKK predicted genes by hadthe Multiple between Expectation one and nine Maximization exons, with for seven Motif ElucidationBnaMKK genes (MEME) (BnaMKK4 suite. The, BnaMKK5 number, BnaMKK8.1of discoverable, BnaMKK8.2 motifs was, BnaMKK9.1 set to ten, BnaMKK9.2 for both BnaMKKs, and BnaMKK10 and BnaMAPKs.) consisting Motifof a single 4 in andBnaMKK very long proteins exon, is much only longer present than in the any PLN00034 other exon superfamily in the other ofBnaMKK proteins genes and defines(Figure 4a).bona The fide number MKKs; of another exons motifamong presents BnaMAPK in both genes BnaMKKs varied from and BnaMAPKstwo to 13, with was three related C groupto the catalyticmembers domain (BnaMAPK1 of serine/threonine-specific, BnaMAPK2, and BnaMAPK14 and tyrosine-specific) having only two protein exons kinases (Fig- ure(PKc-like) 4b). (Figure5). All BnaMKK family members shared motifs 1, 4, 6, and 9, while motifsThen, 2, 5, the and conserved 7 were shared motifs among of BnaMKK all BnaMKK and BnaMAPK members proteins with the were sole predicted exception by of theBnaMKK10. Multiple MotifExpectation 10 was Maximization only identified for in Motif BnaMKK3.1 Elucidation and BnaMKK3 (MEME) suite. (Figure The5a). number From ofa similar discoverable analysis motifs on BnaMAPK was set family to ten members, for both BnaM motifsKKs 11, 13, and 14, BnaMAPKs. 15, 16, 18, and Motif 19 were 4 in BnaMKKdetected in proteins all BnaMAPK is only members, present in with the motifPLN00034 18 in Dsuperfamily group BnaMKKs of proteins located and C-terminal defines bonato motif fide 17, MKKs; and motifs another 11, motif 15, 16, present and 19s in located both BnaMKKs between motif and BnaMAPKs 13 and motif was 14 inrelated each toprotein the catalytic (Figure 5domainb). The of clear serine/threonine divergence in-specificBnaMKK andand tyrosiBnaMAPKne-specificgene protein structures kinases and (PKcmotif-like) composition (Figure 5). of All their BnaMKK encoded family proteins members may indicate sharedfunctional motifs 1, 4, differentiation. 6, and 9, while motifs 2, 5, and 7 were shared among all BnaMKK members with the sole exception of BnaMKK10. Motif 10 was only identified in BnaMKK3.1 and BnaMKK3 (Figure 5a). From a similar analysis on BnaMAPK family members, motifs 11, 13, 14, 15, 16, 18, and 19 were

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detected in all BnaMAPK members, with motif 18 in D group BnaMKKs located C-termi- Int. J. Mol. Sci. 2021, 22, 544 nal to motif 17, and motifs 11, 15, 16, and 19 located between motif 13 and motif 14 in7 each of 21 protein (Figure 5b). The clear divergence in BnaMKK and BnaMAPK gene structures and motif composition of their encoded proteins may indicate functional differentiation.

Figure 4.4. GeneGene structuresstructures of ofBnaMKK BnaMKKand andBnaMAPK BnaMAPKgenes. genes. (a) Exon–intron(a) Exon–intron organization organization of BnaMKK of BnaMKKgenes. genes. (b) Exon–intron (b) Exon– organizationintron organization of BnaMAPK of BnaMAPKgenes. The genes. untranslated The untranslated regions (UTRs)regions are (UTRs) shown are as greenshown blocks, as green exons blocks, as yellow exons blocks, as yellow and blocks, and introns as thin black lines. introns as thin black lines.

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Figure 5. Distribution of conserved motifs in BnaMKK and BnaMAPK proteins. ( (aa)) Conserved Conserved motifs motifs of of BnaMKKs. ( (bb)) Conserved motifs of BnaMAPKs. Each motif is represented by rectangular boxes with different colors. Conserved motifs of BnaMAPKs. Each motif is represented by rectangular boxes with different colors.

2.4. Expression Patterns of BnaMKK and BnaMAPK Genes in Different Tissues and in Re- sponse2.4. Expression to Abiotic Patterns Stress of BnaMKK and BnaMAPK Genes in Different Tissues and in Response to Abiotic Stress Transcriptome deep sequencing (RNA-seq) dataset from the B. napus cultivar Zhongshuang11Transcriptome in public deep sequencing database [38] (RNA-seq) was downloaded dataset from to characterize the B. napus cultivarthe expression Zhong- profilesshuang11 of inBnaMKK public database and BnaMAPK [38] was genes downloaded in different to characterize tissues: Hypocotyls, the expression cotyledo proﬁlesns, roots,of BnaMKK stems,and youngBnaMAPK leaves, genesmature in leaves, different buds, tissues: petals, Hypocotyls, pistils, stamens, cotyledons, anthers, roots, the stems, top ofyoung inflorescences, leaves, mature seeds, leaves, embryos, buds, petals,the seed pistils, coat, stamens,and silique anthers, pericarp the toptissues. of inﬂorescences, Expression (inseeds, Fragments embryos, Per the Kilobase seed coat, of transcript and silique per pericarp Million tissues.mapped Expression reads (FPKM)) (in Fragments was normal- Per Kilobase of transcript per Million mapped reads (FPKM)) was normalized as log (FPKM ized as log2(FPKM + 1) prior to visualization as heatmaps, indicating that most BnaMKK2

Int. J. Mol. Sci. 2021, 22, 544 9 of 21

+ 1) prior to visualization as heatmaps, indicating that most BnaMKK and BnaMAPK genes were differentially expressed in distinct tissues and at various developmental stages (Figure6). Indeed, BnaMKK1, BnaMKK2, BnaMKK3, and BnaMKK9 were consistently expressed at high levels in most tissues, while BnaMKK4, BnaMKK5, BnaMKK6, BnaMKK8, and BnaMKK10 showed little expression. Notably, BnaMKK2.1, BnaMKK2.2, BnaMKK9.1, and BnaMKK9.2 were highly expressed in roots; BnaMKK3.1, BnaMKK3.2, BnaMKK5, and BnaMKK9.2 in anther; BnaMKK2.1, BnaMKK2.2, BnaMKK4, BnaMKK9.1, and BnaMKK9.2 in silique pericarp (Figure6a). Likewise, BnaMAPK1, BnaMAPK2, BnaMAPK13, BnaMAPK14, and BnaMAPK15 were expressed at low levels in most tissues. And our data also showed that BnaMAPK4.1 was highly expressed in roots, stems, leaves, petals, pistils, stamens, and top of inflorescence. In addition, some genes were highly expressed in stamens and anthers, such as BnaMAPK6, BnaMAPK8.1, BnaMAPK10.1, BnaMAPK10.2, BnaMAPK17.1, BnaMAPK19.1, BnaMAPK19.2, BnaMAPK19.3, BnaMAPK20.1, and BnaMAPK20.2 (Figure6b) . These data suggest that BnaMKK and BnaMAPK genes are largely ubiquitously expressed, but not followed the same expression patterns in distinct tissues and developmental stages, which would cause diverse pathways to involve in multiple processes during plant growth and development. To assess the possible contribution of BnaMKK and BnaMAPK genes to abiotic stress responses, we next subjected 3–4-week-old plants from the B. napus cultivar Zhongyou821 to cold, drought, heat, and shading stresses and generated a new RNA-seq dataset. Several BnaMKK and BnaMAPK genes responded to cold, drought, heat, and/or shading stresses (Figure7). For example, BnaMKK1.1/1.2/4/9.1/9.2 expression was highly upregulated by cold stress, BnaMKK9.1 expression by drought stress, and BnaMKK9.1/9.2 expression by shading stress (Figure7a). Likewise, BnaMAPK7.1/7.2/17.2/18.1/18.2/19.1/19.3 were highly induced in response to cold stress, BnaMAPK7.1/7.2/17.2/18.1/18.2 to drought stress, BnaMAPK16/17.2/18.2/18.3 to heat stress, and BnaMAPK7.1/7.2/18.2/18.3 to shading stress (Figure7b). The same stresses also represent the expression of a number of genes: BnaMKK3.2 and BnaMKK9.2 were strongly downregulated by heat stress (Figure7a) , while BnaMAPK18.1 was significantly downregulated by shading (Figure7b). These data demonstrate that BnaMKK genes are sensitive to cold stress at the transcriptional level, while BnaMAPK18.2/18.3 genes are sensitive to cold, drought, heat, and shading stresses. Based on their distinct transcriptional pattern, we hypothesize that BnaMAPK7.1/7.2 play a role in cold, drought, and shading responses but not in heat stress. Similarly, BnaMAPK17.2 transcript levels responded to cold, drought, and heat stresses but more weakly to shading stress, while the expression levels of BnaMAPK17.1 showed no detectable or no significant change. Taken together, BnaMKK and BnaMAPK genes appear to exhibit preferential expression patterns in response to cold, drought, heat, and shading stresses.

2.5. Analysis of Interactions between BnaMKK and C Group BnaMAPK Proteins in Y2H Assays To validate the BnaMKK genes identified by database search, gene-specific primers were independently designed based on the predicted B. napus genes to amplify the coding sequences of BnaMKKs from first-strand cDNAs and confirmed their sequences by DNA sequencing. All clones resolved into 14 BnaMKK independent cDNAs, with two copies each for BnaMKK1 (BnaMKK1.1 and BnaMKK1.2), BnaMKK2, BnaMKK3, BnaMKK5, and BnaMKK8, and one copy each for BnaMKK4, BnaMKK6, BnaMKK9, and BnaMKK10. The length of these sequences varied from 741 bp (BnaMKK10) to 1509 bp (BnaMKK3.1/3.2) (with a mean of 1074.4 bp), encoding proteins of predicted MW from 27.3 kDa (BnaMKK10) to 57.7 kDa (BnaMKK3.2) (with a mean of 40.0 kDa), and a pI ranging from 5.4 (BnaMKK3.1) to 9.0 (BnaMKK4) (with a mean of 6.4) (Table S3). Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 9 of 22

and BnaMAPK genes were differentially expressed in distinct tissues and at various developmental stages (Figure 6). Indeed, BnaMKK1, BnaMKK2, BnaMKK3, and BnaMKK9 were consistently expressed at high levels in most tissues, while BnaMKK4, BnaMKK5, BnaMKK6, BnaMKK8, and BnaMKK10 showed little expression. Notably, BnaMKK2.1, BnaMKK2.2, BnaMKK9.1, and BnaMKK9.2 were highly expressed in roots; BnaMKK3.1, BnaMKK3.2, BnaMKK5, and BnaMKK9.2 in anther; BnaMKK2.1, BnaMKK2.2, BnaMKK4, BnaMKK9.1, and BnaMKK9.2 in silique pericarp (Figure 6a). Likewise, BnaMAPK1, BnaMAPK2, BnaMAPK13, BnaMAPK14, and BnaMAPK15 were expressed at low levels in most tissues. And our data also showed that BnaMAPK4.1 was highly expressed in roots, stems, leaves, petals, pistils, stamens, and top of inflorescence. In addition, some genes were highly expressed in stamens and anthers, such as BnaMAPK6, BnaMAPK8.1, BnaMAPK10.1, BnaMAPK10.2, BnaMAPK17.1, BnaMAPK19.1, BnaMAPK19.2, BnaMAPK19.3, BnaMAPK20.1, and BnaMAPK20.2 (Figure 6b). These data suggest that Int. J. Mol. Sci. 2021, 22, 544 BnaMKK and BnaMAPK genes are largely ubiquitously expressed, but not followed10 ofthe 21 same expression patterns in distinct tissues and developmental stages, which would cause diverse pathways to involve in multiple processes during plant growth and development.

FigureFigure 6. 6. SpatialSpatial and and temporal temporal expression expression pattern pattern of of BnaMKKBnaMKK andand BnaMAPKBnaMAPK genes.genes. ( (aa)) Heatmap Heatmap of of the the expression expression profiles profiles ofof BnaMKKBnaMKK genesgenes in in different different tissues tissues and and developmental developmental stages. stages. ( (bb)) Heatmap Heatmap of of the the expression expression profiles profiles of of BnaMAPKBnaMAPK genesgenes in different tissues and developmental stages. Hy_48h, hypocotyl 48 h after germination; Co_48h, cotyledon 48 h after in different tissues and developmental stages. Hy_48h, hypocotyl 48 h after germination; Co_48h, cotyledon 48 h after germination; Ro_b, root at the bolting stage; Ro_i, root at the initial flowering stage; Ro_f, root at the flowering stage; St_b, germination; Ro_b, root at the bolting stage; Ro_i, root at the initial flowering stage; Ro_f, root at the flowering stage; St_b, stem at the bolting stage; St_i, stem at the initial flowering stage; St_f, stem at the flowering stage; LeY_b, young leaf at the boltingstem at stage; the bolting LeY_i, stage; young St_i, leaf stem at the at theinitial initial flowering flowering stage; stage; LeY_f, St_f, young stem atleaf the at floweringthe flowering stage; stage; LeY_b, LeO young_b, mature leaf at leaf the atbolting the bolting stage; stage; LeY_i, LeO_i, young mature leaf at the leaf initial at the flowering initial flowering stage; LeY_f, stage; young LeO_f, leaf mature at the leaf flowering at the flowering stage; LeO_b, stage; mature Bu_b, leaf bud at atthe the bolting bolting stage; stage; LeO_i, Pe_i, mature petal leafat the at theinitial initial flowering flowering stage; stage; Pe_f, LeO_f, petal mature at the leaf flowering at the flowering stage; Pi_i, stage; pistil Bu_b, at the bud initial at the floweringbolting stage; stage; Pe_i, Pi_f, petal pistil at at the the initial flowering flowering stage; stage; Sta_i,Pe_f, stamen petal at the at the initial flowering flowering stage; stage; Pi_i, Sta_f, pistil stamen at the initialat the flow floweringering stage;stage; At_i, Pi_f, pistilanther at at the the flowering initial flowering stage; Sta_i, stage; stamen At_f, at anther the initial at the flowering flowering stage; stage; Sta_f, IT_i, stamen tip of at main the flowering inflorescence stage; at At_i, the initialanther flowering at the initial stage; flowering IT_f, tip stage; of main At_f, inflorescence anther at the floweringat the flowering stage; IT_i, stage; tip Se_21d, of main inflorescenceseed 21 d after at theflowering; initial flowering Se_40d, stage; IT_f, tip of main inflorescence at the flowering stage; Se_21d, seed 21 days after flowering; Se_40d, seed 40 days after flowering; Em_21d, embryo 21 days after flowering; Em_40d, embryo 40 days after flowering; SC_21d, seed coat 21 days after flowering; SC_40d, seed coat 40 days after flowering; SP_21d, silique pericarp 21 days after flowering; SP_40d, silique

pericarp 40 days after ﬂowering. As much less is known about C group BnaMAPKs relative to the other groups, the coding sequences for BnaMAPK1, BnaMAPK2, and BnaMAPK7 were cloned (the coding sequence for BnaMAPK14 was attempted to clone, but failed). Then, BnaMKK proteins were fused to the GAL4 activation domain and BnaMAPK proteins to the GAL4- binding domain in Y2H vectors. After co-transformation into yeast (Saccharomyces cerevisiae) cells, transformants were selected on SD−Leu−Trp medium and interactions were tested by growth on SD−Ade−His−Leu−Trp medium. As shown in Figure8, BnaMKK3.1, BnaMKK3.2 and BnaMKK9 interacted with BnaMAPK1, BnaMAPK2, and BnaMAPK7. By contrast, BnaMKK1.2 and BnaMKK4 showed no interaction with any of the C group BnaMAPK proteins tested here. BnaMKK8.1, but not its sister protein BnaMKK8.2, showed a strong interaction with BnaMAPK7 (Figure8c). Notably, with the exception of BnaMKK1.2 and BnaMKK4, all other BnaMKKs proteins interacted with BnaMAPK1 (Figure8a). Since Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 10 of 22

seed 40 d after flowering; Em_21d, embryo 21 d after flowering; Em_40d, embryo 40 d after flowering; SC_21d, seed coat 21 d after flowering; SC_40d, seed coat 40 d after flowering; SP_21d, silique pericarp 21 d after flowering; SP_40d, silique pericarp 40 d after flowering.

To assess the possible contribution of BnaMKK and BnaMAPK genes to abiotic stress responses, we next subjected 3–4-week-old plants from the B. napus cultivar Zhongyou821 to cold, drought, heat, and shading stresses and generated a new RNA-seq dataset. Sev- eral BnaMKK and BnaMAPK genes responded to cold, drought, heat, and/or shading stresses (Figure 7). For example, BnaMKK1.1/1.2/4/9.1/9.2 expression was highly upregulated by cold stress, BnaMKK9.1 expression by drought stress, and BnaMKK9.1/9.2 expression by shading stress (Figure 7a). Likewise, BnaMAPK7.1/7.2/17.2/18.1/18.2/19.1/19.3 were highly induced in response to cold stress, BnaMAPK7.1/7.2/17.2/18.1/18.2 to drought stress, BnaMAPK16/17.2/18.2/18.3 to heat stress, and BnaMAPK7.1/7.2/18.2/18.3 to shading

Int. J. Mol. Sci. 2021, 22, 544 stress (Figure 7b). The same stresses also represent the expression of a number of genes:11 of 21 BnaMKK3.2 and BnaMKK9.2 were strongly downregulated by heat stress (Figure 7a), while BnaMAPK18.1 was significantly downregulated by shading (Figure 7b). These data demonstrate that BnaMKK genes are sensitive to cold stress at the transcriptional level, whileABA BnaMAPK18.2 activates several/18.3 MKK-MAPK genes are sensitive signaling to modulescold, drought, directly, heat, the and effect shading of adding stresses. 20 µM − − − − BasedABA on to their the yeast distinct growth transcriptional medium (SD pattern,Ade Hiswe hypothesizeLeu Trp medium) that BnaMAPK7.1 were tested./ However,7.2 play Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 11 of 22 a most role interactions in cold, drought, were not and affected shading by the responses presence ofbut ABA, not suggesting in heat stress. that external Similarly, ABA BnaMAPK17.2is unlikely to transcript directly activate levels responded BnaMAPKs to via cold, BnaMKKs drought, (Figure and heat S1). stresses From these but more results, weaklyBnaMAPK1 to shading appear stress, to be while more the heavily expression connected levels to of upstream BnaMAPK MKKs17.1 showed than BnaMAPK2 no detect- or ableBnaMAPK7.length or no of significant these In addition,sequences change. because varied Taken BnaMKK9 from together, 741 andbp BnaMKK ( BnaMKK3BnaMKK10 and interacted)BnaMAPK to 1509 bp with genes (BnaMKK3.1 the appear same set /to3.2 of) exhibitpartners(with preferentiala mean here, of these 1074.4 expression two bp), MKK encoding patterns proteins proteins in may response play of predicted a similarto cold, roleMW drought, in from linking 27.3 heat, distinctkDa and (BnaMKK10) shading signaling stresses.pathwaysto 57.7 kDa converging (BnaMKK3.2) from separate (with a MAPK mean proteins. of 40.0 kDa), and a pI ranging from 5.4 (BnaMKK3.1) to 9.0 (BnaMKK4) (with a mean of 6.4) (Table S3). As much less is known about C group BnaMAPKs relative to the other groups, the coding sequences for BnaMAPK1, BnaMAPK2, and BnaMAPK7 were cloned (the coding sequence for BnaMAPK14 was attempted to clone, but failed). Then, BnaMKK proteins were fused to the GAL4 activation domain and BnaMAPK proteins to the GAL4-binding domain in Y2H vectors. After co-transformation into yeast (Saccharomyces cerevisiae) cells, transformants were selected on SD−Leu−Trp medium and interactions were tested by growth on SD−Ade−His−Leu−Trp medium. As shown in Figure 8, BnaMKK3.1, BnaMKK3.2 and BnaMKK9 interacted with BnaMAPK1, BnaMAPK2, and BnaMAPK7. By contrast, BnaMKK1.2 and BnaMKK4 showed no interaction with any of the C group BnaMAPK proteins tested here. BnaMKK8.1, but not its sister protein BnaMKK8.2, showed a strong interaction with BnaMAPK7 (Figure 8c). Notably, with the exception of BnaMKK1.2 and BnaMKK4, all other BnaMKKs proteins interacted with BnaMAPK1 (Figure 8a). Since ABA activates several MKK-MAPK signaling modules directly, the effect of adding 20 µM ABA to the yeast growth medium (SD−Ade−His−Leu−Trp medium) were tested. However, most interactions were not affected by the presence of ABA, suggesting that external ABA is unlikely to directly activate BnaMAPKs via BnaMKKs (Figure S1). From these results, BnaMAPK1 appear to be more heavily connected to upstream MKKs than BnaMAPK2 or FigureFigure 7. 7.HeatmapHeatmap representation representation of ofBnaMKKBnaMKK andand BnaMAPKBnaMAPK genegene expression expression patterns patterns upon upon exposure exposure to tocold, cold, drought, drought, BnaMAPK7. In addition, because BnaMKK9 and BnaMKK3 interacted with the same set heat,heat, or orshading shading stress. stress. (a) ( aExpression) Expression profiles proﬁles of ofBnaMKKBnaMKK genesgenes under under four four stresses stresses treatments. treatments. (b ()b Expression) Expression profiles proﬁles of of of partners here, these two MKK proteins may play a similar role in linking distinct sig- BnaMAPKBnaMAPK genesgenes under under four four stresses stresses treatments. treatments. naling pathways converging from separate MAPK proteins. 2.5. Analysis of Interactions between BnaMKK and C Group BnaMAPK Proteins in Y2H As- says To validate the BnaMKK genes identified by database search, gene-specific primers were independently designed based on the predicted B. napus genes to amplify the coding sequences of BnaMKKs from first-strand cDNAs and confirmed their sequences by DNA sequencing. All clones resolved into 14 BnaMKK independent cDNAs, with two copies each for BnaMKK1 (BnaMKK1.1 and BnaMKK1.2), BnaMKK2, BnaMKK3, BnaMKK5, and BnaMKK8, and one copy each for BnaMKK4, BnaMKK6, BnaMKK9, and BnaMKK10. The

FigureFigure 8.8. Yeast two-hybridtwo-hybrid assaysassays betweenbetween BnaMKKBnaMKK andand CC groupgroup BnaMAPKBnaMAPK proteinsproteins inin thethe Y2HY2H goldgold system.system. ((aa)) ProteinProtein interactioninteraction analysisanalysis betweenbetween BnaMKKBnaMKK familyfamily membersmembers andand BnaMAPK1.BnaMAPK1. ((bb)) ProteinProtein interactioninteraction analysisanalysis betweenbetween BnaMKKBnaMKK familyfamily membersmembers andand BnaMAPK2.BnaMAPK2. ((cc)) ProteinProtein interactioninteraction analysisanalysis betweenbetween BnaMKKBnaMKK familyfamily membersmembers andand BnaMAPK7.BnaMAPK7.

3. Discussion 3. Discussion MAPK modules are major signaling pathways involved in biotic and abiotic stress MAPK modules are major signaling pathways involved in biotic and abiotic stress responses in plants [39]. This signaling pathway bridges external stimuli and cellular re- responses in plants [39]. This signaling pathway bridges external stimuli and cellular sponses and is evolutionarily conserved across eukaryotes [12]. In plants, upstream MAP- KKKs phosphorylate and activate the downstream MKKs, which act as a middle converging point in MAPK cascades. MKKs then activate their own downstream MAPKs to transduce specific stimuli and evoke responses to stresses [5]. The MKK and MAPK gene families have been systematically investigated in Arabidopsis [7], rice (Oryza sativa) [40], maize (Zea mays) [41], wheat (Triticum aestivum) [42], diploid cotton (Gossypium raimondii) [43],

Int. J. Mol. Sci. 2021, 22, 544 12 of 21

responses and is evolutionarily conserved across eukaryotes [12]. In plants, upstream MAP- KKKs phosphorylate and activate the downstream MKKs, which act as a middle converging point in MAPK cascades. MKKs then activate their own downstream MAPKs to transduce speciﬁc stimuli and evoke responses to stresses [5]. The MKK and MAPK gene families have been systematically investigated in Arabidopsis [7], rice (Oryza sativa)[40], maize (Zea mays)[41], wheat (Triticum aestivum)[42], diploid cotton (Gossypium raimondii)[43], barley (Hordeum vulgare)[44], tomato (Solanum lycopersicum)[45], cucumber (Cucumis sativus)[46], and purple false brome (Brachypodium distachyon)[47]. As the number and quality of plant genomes increase, an update on the MKK and MAPK gene families in Brassica species is warranted. In the current study, we carried out a comprehensive genome-wide analysis of the MKK and MAPK complement in ﬁve Brassica species. These results will provide a powerful theoretical foundation for future functional studies.

3.1. Characterization of MKK and MAPK Genes in Brassica Species and Their Evolution In previous studies, BLASTP searches or queries with the keyword “MAPK” were employed to identify and characterize MKK and MAPK genes. Accordingly, 10 MKK and 20 MAPK genes were reported in Arabidopsis [7], eight MKK and 17 MAPK genes in rice [40]; six MKK and 16 MAPK genes in tomato [45]; nine MKK and 19 MAPK genes in maize [41]; six MKK and 14 MAPK genes in cucumber [46]; and 12 MKK and 16 MAPK genes in Brachypodium distachyon [47]. However, in most cases, initial reports of MKK and MAPK gene families in Brassica species were not reliable enough to allow a classification of these gene families. For example, 32 BraMAPK genes were initially identified in B. rapa, but the authors pointed out that 13 out of the 32 original BraMAPK genes in the Brasssica Database BRAD were inaccurate due to various errors [48]. Later, 15 BraMKK genes and 34 BraMAPK genes were reported in B. rapa that took the subgenomes into account [49]. Similarly in B. napus, Liang et al. identified seven BnaMKK genes and 12 BnaMAPK genes using Arabidopsis sequences as queries [50]. In the present study, Arabidopsis protein sequences of AtMKK and AtMAPK families were used to identify the full and up-to date complement of MKK and MAPK proteins in these five Brassica species. More accurate information on the quantity and structure of MKKs and MAPKs have been mined in our work, a total of 11 BjuMKKs and 25 BjuMAPKs from B. juncea, 14 BnaMKKs and 29 BnaMAPKs from B. napus, seven BniMKKs and 18 BniMAPKs from B. nigra, six BolMKKs and six BolMAPKs from B. oleracea, nine BraMKKs and 14 BraMAPKs from B. rapa were identified (Table1). These MKKs and MAPKs were classified into four groups (A–D), as in Arabidopsis [7]. Notably, most MKK and MAPK family members appeared to be conserved in Brassica species, especially in B. juncea and B. napus, whereas multiple members were lost in B. oleracea. In the MKK family, MKK1/4/6 were conserved across all five Brassica species; MKK3/5/10 were conserved in Brassica species, excluding B. oleracea; and MKK8 was conserved in Brassica species but absent in B. nigra. In the MAPK family, no MAPK protein was absolutely conserved across all five Brassica species, but many were shared four out of the five species. For instance, MAPK1/2/9/10/13/15 were missing in B. oleracea; MAPK4/17 were absent in B. nigra; MAPK6 was not detected in B. rapa; MAPK19 was absent in B. juncea (Table1). Unlike in previous report, all B. napus MKK genes existed as single copy and that MKK7, MKK8, and MKK10 had no obvious B. napus orthologs [50]. In our study, 14 BnaMKK genes were identified, including the single copy genes BnaMKK4/6/9/10 and the two copies genes BnaMKK1/2/3/5/8, showing that the number of most BnaMKK and BnaMAPK genes in B. napus has been retained or increased after whole-genome duplication (WGD) event. However, the sequence with high homology to AtMKK7 was failed to identify in B. napus (Table S3), suggesting that B. napus has no clear ortholog for this gene or BnaMKK7 gene may be lost after the WGD event. Furthermore, the phylogenetic trees and motif analysis indicated that BnaMKKs and BnaMAPKs were evolutionarily conservative, but functionally differentiated. Our data suggest that the retained and duplicated MKKs and MAPKs may have different functions during the growth and development process of the Brassica species considered here. Int. J. Mol. Sci. 2021, 22, 544 13 of 21

3.2. Functional Divergence of MKK and MAPK Genes during Growth and Development Increasing evidence support a role for MKKs and MAPKs in plant growth and development [4,51]. For example, HvMKK3 regulated seed dormancy in barley [52], while the Arabidopsis ortholog AtMKK3 influenced blue light-mediated seedling development in Arabidopsis [53]. Our data showed that BnaMKK3.1 and BnaMKK3.2 were highly expressed in different tissues at different stages, including seed germination stages and anther development stages, suggesting that BnaMKK3.1 and BnaMKK3.2 may regulate seed and anther development. In Arabidopsis, MKK9 was reported to control phosphate acquisition [54], nitrogen acquisition, anthocyanin accumulation [55], and leaf development [30]. In our study, BnaMKK9.1 and BnaMKK9.2 were expressed at high levels in young and mature leaves at the bolting, initial flowering and flowering stages, suggesting possible roles in leaf development. MKK4 and MKK5 were reported to modulate stomatal development [56], while MKK6 appears to play an important role in meiotic cytokinesis during pollen development [31]. Here, the putative B. napus orthologs BnaMKK4, BnaMKK5, and BnaMKK6 were seldom expression in most tissues and developmental stages, supporting the notion that these genes may not regulate growth or development or that any such regulation might occur at the post-transcriptional level. AtMKK7 negatively regulates polar auxin transport [57], but the clear ortholog in B. napus have been unable to isolated to date. These data indicated that the loss of some BnaMKKs may lead to the functional redundancy of retained and increased BnaMKKs, enabling plants to maintain normal growth and development process. BnaMAPK genes exhibited distinct expression patterns across different tissues at various stages; indeed, 75.9% of BnaMAPK genes were constitutively expressed at high levels in all tissues and organs, with the remaining genes expressed at low levels in most tested tissues (Figure6). Most research has thus far focused on MAPK3/6 and MAPK4 because of the ease by which their activation can be detected. In Arabidopsis, MAPK3 and its homologous gene MAPK6 were reported to control stomatal development [20,58,59], pollen development [60], anther development [61], and shoot branching [62]. MAPK4 facilitates cell plate expansion and male-specific meiotic cytokinesis [33,63]. Similarly, BnaMAPK4.1, BnaMAPK4.2 and BnaMAPK6 were observed to highly express in all tissues at all stages. Moreover, BnaMAPK10.1 and BnaMAPK10.2 were also highly expressed in different tissues and at different stages, especially in petal, stamen and anther at the initial flowering and flowering stage. These data suggest that A group BnaMAPKs may play a more important regulatory role during growth and development. In addition, MAPK12 was reported to be a novel negative regulator of auxin signaling [64], while MAPK18 mediates cortical microtubule stabilization in Arabidopsis [65]. In the present study, three orthologs for AtMAPK18 in B. napus were identified: BnaMAPK18.2 and BnaMAPK18.3 showed higher expression levels than BnaMAPK18.1 in anther at the initial flowering and flowering stage, which indicated that there may be functional redundancy and functional differentiation among the members of BnaMAPK18. The clear ortholog for AtMAPK12 was not identified, suggesting that MAPK12 function may have been lost and that of MAPK18 expanded in B. napus. The analysis of the tissue- or organ-specific expression patterns of BnaMKK and BnaMAPK genes indicated their functional redundancy and divergence during plant growth and development.

3.3. Functional Divergence of MKK and MAPK Genes in Response to Abiotic Stresses Plants have evolved complex mechanisms to respond to adverse environmental conditions and defend themselves against abiotic stress, including cold, drought, osmolarity, heavy metals, and wounding, as well as biotic stress like pathogen and herbivore attacks [17]. Accumulating evidence has demonstrated that MKK and MAPK genes play key roles in the control of plant responses to biotic- and abiotic stresses and to phyto- hormones [15]. For example, MKK1 mediates salt tolerance in rice [66], cotton [67], and Arabidopsis, and both MKK1 and MKK2 participate in plant responses to salt and cold stress [24] and to pathogen attacks in Arabidopsis [4]. Similarly, in our study, BnaMKK1.1, BnaMKK1.2 and BnaMKK2.1 showed high expression levels when plants were exposed Int. J. Mol. Sci. 2021, 22, 544 14 of 21

to cold stress conditions. Since these three genes also showed high expression levels in growth and development process, we speculated that they play positive roles in the cross-talk between growth and development and abiotic stresses at the transcriptional level. For BnaMKK2.2, it expressed highly in the growth and development process, but not in response to stresses, which proved the functional differentiation of BnaMKKs in B. napus. MKK3 was reported to mediate jasmonate [68] and pathogen signaling [69] in Arabidopsis, and responses to ABA and osmotic stress in maize [70]. Here, BnaMKK3.1 expression responded to shading stress, and BnaMKK3.2 to drought, heat, and shading stresses, although the amplitude of gene expression changes did not rise over 2-fold, sug- gestive of a weak transcriptional response for these two genes under these four stress conditions, possibly pointing to a major role in response to other types of stress. In cotton, MKK4 regulates ABA, gibberellic acid, and hydrogen peroxide signaling [71]. MKK5 itself is involved in responses to salt and drought stress [72] and both MKK4 and MKK5 play a vital role in response to wounding and herbivory attack by promoting ethylene production via the transcriptional upregulation of 1-Aminocyclopropane-1-Carboxylate Synthase in Arabidopsis [73]. By contrast, BnaMKK4 only responded to cold stress, and BnaMKK5 did not respond to any of the stresses tested here, which demonstrated that MKKs genes have different patterns in stress response in different plant species. Furthermore, MKK9 plays a pivotal role in increasing alternative respiration in salt-treated Arabidopsis [74], and is also involved in rehydration-triggered ethylene production in roses (Rosa hybrida)[75]. Here, BnaMKK9.1 and BnaMKK9.2 were found to respond to cold, drought, and shading stress, and BnaMKK9.2 was downregulated under heat stress, suggesting that BnaMKK9.1 and BnaMKK9.2 may play important roles in response to abiotic stress. Therefore, BnaMKK3 and BnaMKK9 may regulate the stress resistance more quickly than other BnaMKKs at the transcriptional level in B. napus. Of the characterized MAPK family members, MAPK3 transcript levels are induced by low temperature, osmotic stress, and mechanical stimuli in Arabidopsis [76], and show a rapid response to salt, heat, waterlogging, and wounding in B. rapa [48], as are the B. rapa genes MAPK4 and MAPK5 [48]. In Arabidopsis, MAPK6 transcript levels are unaffected by low temperature, low humidity, touch, or wounding, whereas MAPK6 kinase activity is rapidly activated [77]. Here, we found no evidence for a B. napus ortholog of AtMAPK3 or AtMAPK5. In addition, BnaMAPK4.1 and BnaMAPK4.2 were expressed at low levels and BnaMAPK6 expression remained constant across all stress treatments. About half of all BnaMKK genes (42.9%) and BnaMAPK genes (48.3%) were induced by cold stress. These numbers varied for BnaMKK expression in response to single stress treatment, but not for BnaMAPK genes, which exhibited close to 50% differentially expressed members: 28.6% BnaMKK and 44.8% BnaMAPK genes were induced by drought stress, 21.4% BnaMKK and 44.8% BnaMAPK genes were induced by heat stress, and 28.6% BnaMKK and 48.3% BnaMAPK genes were induced by shading stress. Furthermore, 7.1% BnaMKK and 17.2% BnaMAPK showed changes in transcript levels in response to all four stress conditions, and 21.4% BnaMKK and 41.4% BnaMAPK genes were responsive to any three stress conditions (Figure7). For instance, BnaMAPK7.1 and BnaMAPK7.2 were expressed at high levels in response to cold, drought, and shading stress, BnaMAPK17.2 was expressed at high levels after cold, drought, and heat treatment, while BnaMAPK17.1 did not respond to any treatment, suggesting that the functions of BnaMAPKs may be redundantly speciﬁed in response to various stress conditions in B. napus. Taken together, these results indicate that BnaMKK and BnaMAPK transcript levels were generally modulated by growth and developmental signals and that the encoded proteins may play an important role in abiotic stresses like cold, drought, heat, and shading. These observations also highlight the possible extent of crosstalk between growth or developmental signals and abiotic stress responses.

3.4. Interaction Analysis between BnaMKKs and C Group BnaMAPKs Plant MAPKs regulate numerous physiological responses by interacting with upstream and downstream protein components. A number of MKK-MAPK interaction Int. J. Mol. Sci. 2021, 22, 544 15 of 21

partners have been characterized from many species, like Arabidopsis [78], rice [79], watermelon (Citrullus lanatus)[80], maize [41], and cotton [43]. The MKK4/5-MAPK3/6 pathway was reported to control stomatal development by phosphorylating the basic helix–loop– helix (bHLH) transcription factor SPEECHLESS [20,58,59]. The MKK7-MAPK3/6 cascade controls shoot branching by phosphorylating the auxin transporter PIN-FORMED 1 [62]. Similarly, the interaction between MKK6 and MAPK4 facilitates male-specific meiotic cytokinesis and root development in Arabidopsis [33,63]. Here, the interactions between BnaMKKs and the MAPKs BnaMAPK1, BnaMAPK2, and BnaMAPK7 were tested by yeast two-hybrid. BnaMAPK1 appeared to be more promiscuous than BnaMAPK2 or BnaMAPK7 based on the number of interactions detected: 12 for BnaMAPK1, three for BnaMAPK2 and four for BnaMAPK7, suggesting that C group BnaMAPK1 may play a more important role than BnaMAPK2 or BnaMAPK7. MKK3 in cotton and Arabidopsis were reported to stimulate the activity of C group MAPKs in response to ABA and pathogen attacks [43,69]. A previous report also determined that the MKK9-MAPK1/2 module mediates SA signaling in the B. napus cultivar DH12075 [50]. Here, our data showed that BnaMKK3.1, BnaMKK3.2, and BnaMKK9 interacted with the C group MAPKs BnaMAPK1/2/7. A functional link suggested by interaction should be reflected in the expression pattern of the encoding genes; indeed, BnaMKK3.1, BnaMKK3.2, and BnaMKK9 exhibited a similar temporal and spatial expression pattern to that of BnaMAPK1. By contrast, the expression of this subset of BnaMKK genes showed contrasting responses to stress, with BnaMKK9 being highly expressed and BnaMKK3.1 and BnaMKK3.2 having a low expression level. We therefore speculate that the BnaMKK3.1/3.2/9-BnaMAPK1 module may be of significance for plant growth and development. BnaMKK9-BnaMAPK1 may also play a major role in response to abiotic stress, while BnaMKK3.1/3.2-BnaMAPK1 may regulate stress responses at the translational or post-translational levels, rather than at the transcriptional level. Moreover, BnaMKK3.1 and BnaMKK3.2 expression were especially highly expressed in anthers, suggesting that the BnaMKK3-BnaMAPK1 module may participate in anther development. Since BnaMKK9 was also highly expressed in all tissues at all stages, especially in silique pericarps, we hypothesize that BnaMKK9 interacting with BnaMAPK2 may positively regulate seed development by controlling photosynthesis and nutrient biosynthesis in siliques. Cotton (Gossypium hirsutum) MAPK7 was reported to regulate plant growth and development [81]. Interestingly, BnaMAPK7 expression was higher than that of BnaMAPK1 or BnaMAPK2 in all conditions tested here, and followed a similar expression pattern as BnaMKK3.1, BnaMKK3.2, and BnaMKK9, suggesting that the BnaMKK3.1/3.2/9-BnaMAPK7 module functions in growth, development, and stress signaling. Therefore, it is very likely that C group BnaMAPKs have redundant functions during these signaling events, in agreement with prior research in Arabidopsis and cotton [69,81]. Besides the BnaMKK3.1/3.2/9-BnaMAPK1/2/7 module, the new interaction pairs BnaMKK8.1-BnaMAPK1/7 were also detected, although BnaMKK8.1 was barely expressed across our two transcriptome datasets, suggesting that the function of the BnaMKK8.1-BnaMAPK1/7 module may not be regulated at the transcriptional level. Taken together, our results indicate the possible conservation of BnaMKK3/9-BnaMAPK1/2/7 cascades in response to growth, development, and abiotic stress. Our data also implied an inherent difference in signaling pathways between B. napus and other plant species, and highlighted the necessity to explore MKKs-MAPKs-mediated signaling pathways for use in genetic investigations and breeding of B. napus.

4. Materials and Methods 4.1. Genome-Wide Identiﬁcation of MKK and MAPK Genes in Brassica To comprehensively identify all MKK and MAPK members, whole-genome data for Arabidopsis thaliana (http://www.arabidopsis.org/) and ﬁve Brassica species (B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa)(http://brassicadb.org/brad/)[82] were downloaded to construct a local protein database. Then, Arabidopsis MKK and MAPK proteins were used as query to search against Brassica proteins using BLASTP [83] with an e-value Int. J. Mol. Sci. 2021, 22, 544 16 of 21

of 1 × 10−5 and minimum identity of 50% as the threshold. The HMMER3.0 program (http://hmmer.org/)[84] was employed to conduct a Hidden Markov Model (HMM) search using the serine/threonine-protein kinase-like domain (PF00069) as query with the e-value threshold of 1 × 10−5. The HMMER hits were then integrated with the BLASTP results and parsed by manual editing to remove redundant sequences. Those proteins dis- playing the consensus sequences as described by Chen et al. [47] were considered potential MKK and MAPK proteins. The theoretical pI and MW of the identiﬁed Brassica MKKs and MAPKs proteins were evaluated using Isoelectric Point Calculation of Proteins and Peptides (IPC) server (http://isoelectric.org/)[85].

4.2. Sequence Alignment, Phylogenetic Analysis, Chromosomal Location, and Gene Structure Construction The full-length protein sequences for all MKK and MAPK proteins from the five Brassica species and Arabidopsis were aligned by the Clustal W program in the MEGA-X software [86] with default settings. The resulting file was used to generate a phylogenetic tree with the NJ method and 1000 bootstrap replicates in MEGA-X. The phylogenetic trees were visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree/). The chromosomal positions of BnaMKK and BnaMAPK genes were provided by the Genoscope database and visualized with TBtools software [87]. For the gene structures analysis, the GTF annotation files for the Brassica genomes being considered here were downloaded and uploaded to TBtools to display gene exon–intron structures. Conserved motifs in BnaMKK and BnaMAPK proteins were analyzed using MEME suite v5.1.1 (http://meme- suite.org/tools/meme)[88] with the following optimized parameters: repetition number, any; minimum motif width, 6; maximum motif width, 50; maximum number of motifs, 10. MEME files were visualized with TBtools.

4.3. Plant Material and Growth Conditions The black-seed doubled haploid (DH) inbred B. napus cultivar Zhongyou821 seeds were obtained from the Rapeseed Engineering Research Center of Southwest University in Chongqing, China (CERCR). The healthy seeds were sown in nutritious soil in the growth chamber (Model PGR15, Conviron, Controlled Environments Ltd., Winnipeg, MB, Canada) with 16 h light (25 ◦C)/8 h dark (20 ◦C) photoperiod, 800 µmol·m−2·s−1 light intensity and 75% humidity. Light was provided by a mix of cool white ﬂuorescent tubes (Philips F72T/CW/VHO, Philips Lighting Compant, Somerset, NJ, USA) and Philips 60-W incandescent lamps (Philips Electronics Ltd., Markham, ON, Canada). The photo- synthetically active photon ﬂux density (PPFD) was measured with a quantum LI-185B radiometer/photometer (LI-COR, Inc., Lincoln, NV, USA). Plants were placed on the layer farthest from light banks in the growth chamber. For gene cloning, the leaves, roots and stems were harvested from 3–4-week-old seedlings at four-leaf stage, and frozen immediately in liquid nitrogen, then stored at −80 ◦C until RNA extraction. For RNA-seq analysis, four-leaf seedlings were used for cold, drought, heat, and shading treatments. For cold stress, plants were transferred to a growth chamber set to 4 ◦C and sampled 3 h after transfer. For drought stress, plants were sprayed with 10% PEG 6000 and sampled 3 h later. Heat stress was imposed by transferring plants to a growth chamber set to 42 ◦C; samples were collected 1.5 h later. For the shading treatment, plants were exposed to limited light conditions (300 µmol·m−2·s−1) and samples collected 12 h later. For the controls, seedlings were kept the same growth conditions as before. Samples were immediately frozen in liquid nitrogen and stored at −80 ◦C until sequencing.

4.4. Expression Analyses of the Two Families Genes in Rapeseed Using RNA-Seq Data The RNA-seq dataset of B. napus cultivar Zhongshuang11 (BioProject ID: PRJNA358784) was described previously [38] and covers hypocotyl, cotyledon, root, stem, young leaf, mature leaf, bud, petal, pistil, stamen, anther, top of inﬂorescence, seed, embryo, seed coat, and silique pericarp at multiple developmental stages. Expression values (in Frag- ments Per Kilobase of transcript per Million mapped reads (FPKM)) of all BnaMKK and Int. J. Mol. Sci. 2021, 22, 544 17 of 21

BnaMAPK genes were then normalized as log2(FPKM + 1) and submitted the results to TBtools to generate a heatmap. For RNA-seq analysis of stress responses, equal amounts of three individual plants leaves (by weight) were pooled as one biological replicate, and three independent biological replicates for each treatment and control were used in this study. Libraries were constructed using the NEBnext ultra RNA library prep kit (Illumina, San Diego, CA, USA) and sequenced at Beijing BioMarker Bioinformatics Technology Co., Ltd. (Beijing, China) on an Illumina platform. Raw RNA-seq data have been de- posited at the Short Read Archive (SRA) repository (BioProject ID: PRJNA680826). The log2 (Fold-Change) values were calculated and submitted to TBtools to generate heatmaps. In all heatmaps, red and blue color represent increased or decreased expression levels, respectively, in comparison to controls; gray color indicates genes whose expression values were not signiﬁcantly different from the controls (false discovery rate > 0.05).

4.5. Total RNA Isolation and cDNA Synthesis Total RNAs from seedlings leaves, roots and stems tissues were respectively isolated using the RNAprep pure plant kit (Tiangen, Beijing, China) according to the manufacturer’s instructions. The RNA concentration and quality were determined on a Nanodrop 2000c spectrophotometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA). One microgram of high- quality RNA was used for ﬁrst-strand cDNA synthesis with the primescript RT reagent kit (Takara Bio, Mountain View, CA, USA), equal amounts (by volume) cDNA of each tissues, including leaves, roots and stems, were pooled as the mixture cDNA sample for gene cloning. 18S primers were used to assess the quality of the cDNA preparations. Primer sequences are listed in Table S4.

4.6. Gene Cloning, Plasmid Construction, and Yeast Two-Hybrid Assays The open reading frames (ORFs) for BnaMKK and C group BnaMAPK members were amplified with gene-specific primers (Table S4) from Zhongyou821 first-strand cDNAs and ligated into pGEM-T easy vector (Promega, Madison, WI, USA) following the manufacturer’s instructions. After sequencing and alignment against the reference genome, the confirmed BnaMKK ORFs were individually cloned into the pGADT7 prey vector, and C group BnaMAPK ORFs into the pGBKT7 bait vector. Yeast two-hybrid (Y2H) assays were performed using the Matchmaker gold Y2H system (Clontech/Takara Bio, Mountain View, CA, USA), following the manufacturer’s protocol for co-transformation. The transformants were plated on SD–Leu–Trp medium and grown for 2–4 days at 30 ◦C, then transferred onto solid SD–Ade–His–Leu–Trp medium and grown for 3–5 days at 30 ◦C to observe protein interactions. pGBKT7-53 and pGADT7- T were used as positive controls, pGBKT7-Lam and pGADT7-T were used as negative controls. To determine the intensity of interaction between bait and prey, yeast cultures at stationary phase were diluted to 1:10, 1:100 and 1:1000 and spotted onto selective medium. The effect of ABA on BnaMKK-BnaMAPK interactions were also tested by adding 20 µM ABA to the selective SD–Ade–His–Leu–Trp medium and spotting yeast cultures at stationary phase diluted 1:1000.

5. Conclusions Our work identified 47 MKK and 92 MAPK genes from five Brassica species (B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa), with 14 BnaMKK and 29 BnaMAPK coming from B. napus. Multiple protein alignments and phylogenetic analysis of Arabidopsis and Brassica MKKs and MAPKs helped classified them into 4 subgroups. Also, their chromosomal locations, exon–intron structure and conserved protein motifs were characterized. Our results suggest that genome duplication contributed to the expansion of the BnaMKK and BnaMAPK gene families. In the present study, we focused on the expression patterns of BnaMKK and BnaMAPK genes in different tissues and at developmental stages, as well as upon exposure to cold, drought, heat, or shading stress, revealing that BnaMKK and BnaMAPK genes may play key roles in growth and development and responses to Int. J. Mol. Sci. 2021, 22, 544 18 of 21

environmental stress. Finally, 12 BnaMKKs-BnaMAPK1, 3 BnaMKKs-BnaMAPK2, and 4 BnaMKKs-BnaMAPK7 interactions were deﬁned by yeast two-hybrid, of which the BnaMKK3.1/3.2/9-BnaMAPK1/2/7 module may play a conserved and key role during growth, development, and stress response. It will be interesting to explore their functions during abiotic and biotic stress responses.

Supplementary Materials: Supplementary materials can be found at https://www.mdpi.com/1422 -0067/22/2/544/s1. Table S1: List of MKK and MAPK genes identified in A. thaliana and Brassica genomes; Table S2: Sequence length, molecular weight, and isoelectric point information for BnaMKK and BnaMAPK proteins predicted in B. napus; Table S3: Sequence length, molecular weight, and isoelectric point information for BnaMKK and C group BnaMAPK proteins cloned in B. napus; Table S4: Primers used to amplify and detect BnaMKK and C group BnaMAPK genes in this study. Figure S1, Yeast two-hybrid assays between BnaMKK and C group BnaMAPK proteins in the presence of 20 µM ABA. (a) Protein interaction analysis between BnaMKK family members and BnaMAPK1 with ABA. (b) Protein interaction analysis between BnaMKK family members and BnaMAPK2 with ABA. (c) Protein interaction analysis between BnaMKK family members and BnaMAPK7 with ABA. Author Contributions: C.Q. and Y.L. conceived and designed the study; Z.W., Y.W. and X.M. performed the bioinformatics analyses; Z.W., X.M., X.Z. and M.Y. performed the yeast two-hybrid assays, W.M., D.Z. and D.Y. helped to prepare figures and tables; Z.W. wrote the manuscript; K.L. and J.L. reviewed the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This research is funded by the National Natural Science Foundation of China (31271756 and 31872876), the Fundamental Research Funds for the Central Universities (XDJK2020D023), and the 111 Project of China (B12006). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Not applicable. Acknowledgments: We deeply thank Jiaming Yin from Southwest University (China) for providing the Zhongyou821 DH line seeds. We also thank Liyuan Zhang, Bo Yang, and Wei Chang from Southwest University (China) for their contributions in performing the data analyses. Conflicts of Interest: The authors declare no conflict of interest.

References 1. Colcombet, J.; Hirt, H. Arabidopsis MAPKs: A complex signalling network involved in multiple biological processes. Biochem. J. 2008, 413, 217–226. [CrossRef][PubMed] 2. Kelkar, N.; Gupta, S.; Dickens, M.; Davis, R.J. Interaction of a mitogen-activated protein kinase signaling module with the neuronal protein JIP3. Mol. Cell. Biol. 2000, 20, 1030–1043. [CrossRef][PubMed] 3. Jonak, C.; Ligterink, W.; Hirt, H. MAP kinases in plant signal transduction. Cell. Mol. Life Sci. 1999, 55, 204–213. [CrossRef] [PubMed] 4. Suarez-Rodriguez, M.C.; Petersen, M.; Mundy, J. Mitogen-Activated Protein Kinase Signaling in Plants. Annu. Rev. Plant Biol. 2010, 61, 621–649. [CrossRef] 5. Sinha, A.K.; Jaggi, M.; Raghuram, B.; Tuteja, N. Mitogen-activated protein kinase signaling in plants under abiotic stress. Plant Signal. Behav. 2011, 6, 196–203. [CrossRef][PubMed] 6. Zhang, S.; Klessig, D.F. MAPK cascades in plant defense signaling. Trends Plant Sci. 2001, 6, 520–527. [CrossRef] 7. Ichimura, K.; Shinozaki, K.; Tena, G.; Sheen, J.; Henry, Y.; Champion, A.; Kreis, M.; Zhang, S.; Hirt, H.; Wilson, C.; et al. Mitogen-activated protein kinase cascades in plants: A new nomenclature. Trends Plant Sci. 2002, 7, 301–308. [CrossRef] 8. Pitzschke, A.; Schikora, A.; Hirt, H. MAPK cascade signalling networks in plant defence. Curr. Opin. Plant Biol. 2009, 12, 421–426. [CrossRef] 9. Neill, S.; Desikan, R.; Hancock, J. Hydrogen peroxide signalling. Curr. Opin. Plant Biol. 2002, 5, 388–395. [CrossRef] 10. Popescu, S.; Popescu, G.; Bachan, S.; Zhang, Z.; Gerstein, M.; Snyder, M.; Dinesh-kumar, S.P. MAPK target networks in Arabidopsis thaliana revealed using functional protein microarrays. Genes Dev. 2009, 23, 80–92. [CrossRef][PubMed] 11. Taj, G.; Agarwal, P.; Grant, M.; Kumar, A. MAPK machinery in plants: Recognition and response to different stresses through multiple signal transduction pathways. Plant Signal. Behav. 2010, 5, 1370–1378. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 544 19 of 21

12. Jonak, C.; Ökrész, L.; Bögre, L.; Hirt, H. Complexity, cross talk and integration of plant MAP kinase signalling. Curr. Opin. Plant Biol. 2002, 5, 415–424. [CrossRef] 13. Hamel, L.P.; Nicole, M.C.; Sritubtim, S.; Morency, M.J.; Ellis, M.; Ehlting, J.; Beaudoin, N.; Barbazuk, B.; Klessig, D.; Lee, J.; et al. Ancient signals: Comparative genomics of plant MAPK and MAPKK gene families. Trends Plant Sci. 2006, 11, 192–198. [CrossRef] [PubMed] 14. Moustafa, K.; AbuQamar, S.; Jarrar, M.; Al-Rajab, A.J.; Trémouillaux-Guiller, J. MAPK cascades and major abiotic stresses. Plant Cell Rep. 2014, 33, 1217–1225. [CrossRef][PubMed] 15. Zhang, M.; Su, J.; Zhang, Y.; Xu, J.; Zhang, S. Conveying endogenous and exogenous signals: MAPK cascades in plant growth and defense. Curr. Opin. Plant Biol. 2018, 45, 1–10. [CrossRef] 16. Chen, R.E.; Thorner, J. Function and regulation in MAPK signaling pathways: Lessons learned from the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 2007, 1773, 1311–1340. [CrossRef] 17. Smékalová, V.; Doskoˇcilová, A.; Komis, G.; Šamaj, J. Crosstalk between secondary messengers, hormones and MAPK modules during abiotic stress signalling in plants. Biotechnol. Adv. 2014, 32, 2–11. [CrossRef] 18. Asai, T.; Tena, G.; Plotnikova, J.; Willmann, M.R.; Chiu, W.L.; Gomez-Gomez, L.; Boller, T.; Ausubel, F.M.; Sheen, J. Map kinase signalling cascade in Arabidopsis innate immunity. Nature 2002, 415, 977–983. [CrossRef] 19. Galletti, R.; Ferrari, S.; de Lorenzo, G. Arabidopsis MPK3 and MPK6 play different roles in basal and oligogalacturonide-or flagellin-induced resistance against Botrytis cinerea. Plant Physiol. 2011, 157, 804–814. [CrossRef] 20. Wang, H.; Ngwenyama, N.; Liu, Y.; Walker, J.C.; Zhang, S. Stomatal development and patterning are regulated by environmentally responsive mitogen-activated protein kinases in Arabidopsis. Plant Cell 2007, 19, 63–73. [CrossRef] 21. Eckardt, N.A. A Complete MAPK Signaling Cascade That Functions in Stomatal Development and Patterning in Arabidopsis. Plant Cell 2007, 19, 7. [CrossRef] 22. Gudesblat, G.E.; Iusem, N.D.; Morris, P.C. Guard cell-specific inhibition of Arabidopsis MPK3 expression causes abnormal stomatal responses to abscisic acid and hydrogen peroxide. New Phytol. 2007, 173, 713–721. [CrossRef][PubMed] 23. Takahashi, F.; Mizoguchi, T.; Yoshida, R.; Ichimura, K.; Shinozaki, K. Calmodulin-Dependent Activation of MAP Kinase for ROS Homeostasis in Arabidopsis. Mol. Cell 2011, 41, 649–660. [CrossRef][PubMed] 24. Teige, M.; Scheikl, E.; Eulgem, T.; Dóczi, R.; Ichimura, K.; Shinozaki, K.; Dangl, J.L.; Hirt, H. The MKK2 pathway mediates cold and salt stress signaling in Arabidopsis. Mol. Cell 2004, 15, 141–152. [CrossRef][PubMed] 25. Petersen, M.; Brodersen, P.; Naested, H.; Andreasson, E.; Lindhart, U.; Johansen, B.; Nielsen, H.B.; Lacy, M.; Austin, M.J.; Parker, J.E.; et al. Arabidopsis MAP kinase 4 negatively regulates systemic acquired resistance. Cell 2000, 103, 1111–1120. [CrossRef] 26. Qiu, J.L.; Zhou, L.; Yun, B.W.; Nielsen, H.B.; Fiil, B.K.; Petersen, K.; MacKinlay, J.; Loake, G.J.; Mundy, J.; Morris, P.C. Arabidopsis mitogen-activated protein kinase kinases MKK1 and MKK2 have overlapping functions in defense signaling mediated by MEKK1, MPK4, and MKS1. Plant Physiol. 2008, 148, 212–222. [CrossRef] 27. Furuya, T.; Matsuoka, D.; Nanmori, T. Membrane rigidification functions upstream of the MEKK1-MKK2-MPK4 cascade during cold acclimation in Arabidopsis thaliana. FEBS Lett. 2014, 588, 2025–2030. [CrossRef] 28. Xu, J.; Li, Y.; Wang, Y.; Liu, H.; Lei, L.; Yang, H.; Liu, G.; Ren, D. Activation of MAPK kinase 9 induces ethylene and camalexin biosynthesis and enhances sensitivity to salt stress in Arabidopsis. J. Biol. Chem. 2008, 283, 26996–27006. [CrossRef] 29. Yoo, S.; Sheen, J. MAPK signaling in plant hormone ethylene signal transduction. Plant Signal. Behav. 2008, 3, 848–849. [CrossRef] 30. Zhou, C.; Cai, Z.; Guo, Y.; Gan, S. An Arabidopsis mitogen-activated protein kinase cascade, MKK9-MPK6, plays a role in leaf senescence. Plant Physiol. 2009, 150, 167–177. [CrossRef] 31. Beck, M.; Komis, G.; Müller, J.; Menzel, D.; Šamaj, J. Arabidopsis homologs of nucleus- and phragmoplast-localized kinase 2 and 3 and mitogen-activated protein kinase 4 are essentialfor microtubule organization. Plant Cell 2010, 22, 755–771. [CrossRef] [PubMed] 32. Beck, M.; Komis, G.; Ziemann, A.; Menzel, D.; Samaj, J. Mitogen-activated protein kinase 4 is involved in the regulation of mitotic and cytokinetic microtubule transitions in Arabidopsis thaliana. New Phytol. 2011, 189, 1069–1083. [CrossRef][PubMed] 33. Zeng, Q.; Chen, J.G.; Ellis, B.E. AtMPK4 is required for male-specific meiotic cytokinesis in Arabidopsis. Plant J. 2011, 67, 895–906. [CrossRef][PubMed] 34. Takahashi, Y.; Soyano, T.; Kosetsu, K.; Sasabe, M.; MacHida, Y. HINKEL kinesin, ANP MAPKKKs and MKK6/ANQ MAPKK, which phosphorylates and activates MPK4 MAPK, constitute a pathway that is required for cytokinesis in Arabidopsis thaliana. Plant Cell Physiol. 2010, 51, 1766–1776. [CrossRef][PubMed] 35. Danquah, A.; De Zélicourt, A.; Boudsocq, M.; Neubauer, J.; Frei Dit Frey, N.; Leonhardt, N.; Pateyron, S.; Gwinner, F.; Tamby, J.P.; Ortiz-Masia, D.; et al. Identification and characterization of an ABA-activated MAP kinase cascade in Arabidopsis thaliana. Plant J. 2015, 82, 232–244. [CrossRef] 36. Weng, C.M.; Lu, J.X.; Wan, H.F.; Wang, S.W.; Wang, Z.; Lu, K.; Liang, Y. Over-expression of BnMAPK1 in Brassica napus enhances tolerance to drought stress. J. Integr. Agric. 2014, 13, 2407–2415. [CrossRef] 37. Wang, S.W.; Lu, J.X.; Wan, H.F.; Weng, C.M.; Wang, Z.; Li, J.N.; Lu, K.; Liang, Y. Overexpression of BnMAPK1 enhances resistance to Sclerotinia sclerotiorum in Brassica napus. Acta Agron. Sin. 2014, 40, 745–750. [CrossRef] 38. Lu, K.; Wei, L.; Li, X.; Wang, Y.; Wu, J.; Liu, M.; Zhang, C.; Chen, Z.; Xiao, Z.; Jian, H.; et al. Whole-genome resequencing reveals Brassica napus origin and genetic loci involved in its improvement. Nat. Commun. 2019, 10, 1154. [CrossRef] Int. J. Mol. Sci. 2021, 22, 544 20 of 21

39. Tena, G.; Asai, T.; Chiu, W.-L.; Sheen, J. Plant mitogen-activated protein kinase signaling cascades. Curr. Opin. Plant Biol. 2001, 4, 392–400. [CrossRef] 40. Wankhede, D.P.; Misra, M.; Singh, P.; Sinha, A.K. Rice Mitogen Activated Protein Kinase Kinase and Mitogen Activated Protein Kinase Interaction Network Revealed by In-Silico Docking and Yeast Two-Hybrid Approaches. PLoS ONE 2013, 8, e65011. [CrossRef] 41. Kong, X.; Pan, J.; Zhang, D.; Jiang, S.; Cai, G.; Wang, L.; Li, D. Identification of mitogen-activated protein kinase kinase gene family and MKK-MAPK interaction network in maize. Biochem. Biophys. Res. Commun. 2013, 441, 964–969. [CrossRef][PubMed] 42. Zhan, H.; Yue, H.; Zhao, X.; Wang, M.; Song, W.; Nie, X. Genome-wide identification and analysis of MAPK and MAPKK gene families in bread wheat (Triticum aestivum L.). Genes 2017, 8, 284. [CrossRef][PubMed] 43. Zhang, X.; Xu, X.; Yu, Y.; Chen, C.; Wang, J.; Cai, C.; Guo, W. Integration analysis of MKK and MAPK family members highlights potential MAPK signaling modules in cotton. Sci. Rep. 2016, 6, 29781. [CrossRef][PubMed] 44. Cui, L.; Yang, G.; Yan, J.; Pan, Y.; Nie, X. Genome-wide identification, expression profiles and regulatory network of MAPK cascade gene family in barley. BMC Genom. 2019, 20, 750. [CrossRef][PubMed] 45. Wu, J.; Wang, J.; Pan, C.; Guan, X.; Wang, Y.; Liu, S.; He, Y.; Chen, J.; Chen, L.; Lu, G. Genome-wide identification of MAPKK and MAPKKK gene families in tomato and transcriptional profiling analysis during development and stress response. PLoS ONE 2014, 9, e103032. [CrossRef] 46. Wang, J.; Pan, C.; Wang, Y.; Ye, L.; Wu, J.; Chen, L.; Zou, T.; Lu, G. Genome-wide identification of MAPK, MAPKK, and MAPKKK gene families and transcriptional profiling analysis during development and stress response in cucumber. BMC Genom. 2015, 16. [CrossRef] 47. Chen, L.; Hu, W.; Tan, S.; Wang, M.; Ma, Z.; Zhou, S.; Deng, X.; Zhang, Y.; Huang, C.; Yang, G.; et al. Genome-Wide Identification and Analysis of MAPK and MAPKK Gene Families in Brachypodium distachyon. PLoS ONE 2012, 7, e46744. [CrossRef] 48. Lu, K.; Guo, W.; Lu, J.; Yu, H.; Qu, C.; Tang, Z.; Li, J.; Chai, Y.; Liang, Y.; Pandey, G.K. Genome-wide survey and expression profile analysis of the mitogen-activated protein kinase (MAPK) gene family in Brassica rapa. PLoS ONE 2015, 10, e0132051. [CrossRef] 49. Wu, P.; Wang, W.; Li, Y.; Hou, X. Divergent evolutionary patterns of the MAPK cascade genes in Brassica rapa and plant phylogenetics. Hortic. Res. 2017, 4, 17079. [CrossRef] 50. Liang, W.; Yang, B.; Yu, B.J.; Zhou, Z.; Li, C.; Jia, M.; Sun, Y.; Zhang, Y.; Wu, F.; Zhang, H.; et al. Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.). BMC Genom. 2013, 14, 392. [CrossRef] 51. Šamajová, O.; Plíhal, O.; Al-Yousif, M.; Hirt, H.; Šamaj, J. Improvement of stress tolerance in plants by genetic manipulation of mitogen-activated protein kinases. Biotechnol. Adv. 2013, 31, 118–128. [CrossRef][PubMed] 52. Nakamura, S.; Pourkheirandish, M.; Morishige, H.; Kubo, Y.; Nakamura, M.; Ichimura, K.; Seo, S.; Kanamori, H.; Wu, J.; Ando, T.; et al. Mitogen-activated protein kinase kinase 3 regulates seed dormancy in barley. Curr. Biol. 2016, 26, 775–781. [CrossRef] [PubMed] 53. Sethi, V.; Raghuram, B.; Sinha, A.K.; Chattopadhyay, S. A mitogen-activated protein kinase cascade module, MKK3-MPK6 and MYC2, Is involved in blue light-mediated seedling development in Arabidopsis. Plant Cell 2014, 26, 3343–3357. [CrossRef] [PubMed] 54. Lei, L.; Li, Y.; Wang, Q.; Xu, J.; Chen, Y.; Yang, H.; Ren, D. Activation of MKK9-MPK3/MPK6 enhances phosphate acquisition in Arabidopsis thaliana. New Phytol. 2014, 203, 1146–1160. [CrossRef][PubMed] 55. Luo, J.; Wang, X.; Feng, L.; Li, Y.; He, J.X. The mitogen-activated protein kinase kinase 9 (MKK9) modulates nitrogen acquisition and anthocyanin accumulation under nitrogen-limiting condition in Arabidopsis. Biochem. Biophys. Res. Commun. 2017, 487, 539–544. [CrossRef] 56. Sözen, C.; Schenk, S.T.; Boudsocq, M.; Chardin, C.; Almeida-Trapp, M.; Krapp, A.; Hirt, H.; Mithöfer, A.; Colcombet, J. Wounding and insect feeding trigger two independent MAPK pathways with distinct regulation and kinetics. Plant Cell 2020, 32, 1988–2003. [CrossRef] 57. Dai, Y.; Wang, H.; Li, B.; Huang, J.; Liu, X.; Zhou, Y.; Mou, Z.; Li, J. Increased expression of MAP KINASE KINASE7 causes deficiency in polar auxin transport and leads to plant architectural abnormality in Arabidopsis. Plant Cell 2006, 18, 308–320. [CrossRef] 58. Bergmann, D.C.; Lukowitz, W.; Somerville, C.R. Stomatal development and pattern controlled by a MAPKK kinase. Science 2004, 304, 1494–1497. [CrossRef] 59. Lampard, G.R.; MacAlister, C.A.; Bergmann, D.C. Arabidopsis stomatal initiation is controlled by MAPK-mediated regulation of the bHLH SPEECHLESS. Science 2008, 322, 1113–1116. [CrossRef] 60. Guan, Y.; Meng, X.; Khanna, R.; LaMontagne, E.; Liu, Y.; Zhang, S. Phosphorylation of a WRKY Transcription Factor by MAPKs Is Required for Pollen Development and Function in Arabidopsis. PLoS Genet. 2014, 10, e1004384. [CrossRef] 61. Zhao, F.; Zheng, Y.F.; Zeng, T.; Sun, R.; Yang, J.Y.; Li, Y.; Ren, D.T.; Ma, H.; Xu, Z.H.; Bai, S.N. Phosphorylation of SPOROCYTE- LESS/NOZZLE by the MPK3/6 kinase is required for anther development. Plant Physiol. 2017, 173, 2265–2277. [CrossRef] [PubMed] 62. Jia, W.; Li, B.; Li, S.; Liang, Y.; Wu, X.; Ma, M.; Wang, J.; Gao, J.; Cai, Y.; Zhang, Y.; et al. Mitogen-Activated Protein Kinase Cascade MKK7-MPK6 Plays Important Roles in Plant Development and Regulates Shoot Branching by Phosphorylating PIN1 in Arabidopsis. PLoS Biol. 2016, 14, e1002550. [CrossRef][PubMed] Int. J. Mol. Sci. 2021, 22, 544 21 of 21

63. Kosetsu, K.; Matsunaga, S.; Nakagami, H.; Colcombet, J.; Sasabe, M.; Soyano, T.; Takahashi, Y.; Hirt, H.; Machida, Y. The MAP kinase MPK4 Is required for cytokinesis in Arabidopsis thaliana. Plant Cell 2010, 22, 3778–3790. [CrossRef][PubMed] 64. Lee, J.S.; Wang, S.; Sritubtim, S.; Chen, J.G.; Ellis, B.E. Arabidopsis mitogen-activated protein kinase MPK12 interacts with the MAPK phosphatase IBR5 and regulates auxin signaling. Plant J. 2009, 57, 975–985. [CrossRef][PubMed] 65. Walia, A.; Lee, J.S.; Wasteneys, G.; Ellis, B. Arabidopsis mitogen-activated protein kinase MPK18 mediates cortical microtubule functions in plant cells. Plant J. 2009, 59, 565–575. [CrossRef] 66. Wang, F.; Jing, W.; Zhang, W. The mitogen-activated protein kinase cascade MKK1-MPK4 mediates salt signaling in rice. Plant Sci. 2014, 227, 181–189. [CrossRef] 67. Lu, W.; Chu, X.; Li, Y.; Wang, C.; Guo, X. Cotton GhMKK1 Induces the Tolerance of Salt and Drought Stress, and Mediates Defence Responses to Pathogen Infection in Transgenic Nicotiana benthamiana. PLoS ONE 2013, 8.[CrossRef] 68. Takahashi, F.; Yoshida, R.; Ichimura, K.; Mizoguchi, T.; Seo, S.; Yonezawa, M.; Maruyama, K.; Yamaguchi-Shinozaki, K.; Shinozakia, K. The mitogen-activated protein kinase cascade MKK3-MPK6 is an important part of the jasmonate signal transduction pathway in Arabidopsis. Plant Cell 2007, 19, 805–818. [CrossRef] 69. Dóczi, R.; Brader, G.; Pettkó-Szandtner, A.; Rajh, I.; Djamei, A.; Pitzschke, A.; Teige, M.; Hirt, H. The Arabidopsis mitogen-activated protein kinase kinase MKK3 is upstream of group C mitogen-activated protein kinases and participates in pathogen signaling. Plant Cell 2007, 19, 3266–3279. [CrossRef] 70. Zhang, M.; Pan, J.; Kong, X.; Zhou, Y.; Liu, Y.; Sun, L.; Li, D. ZmMKK3, a novel maize group B mitogen-activated protein kinase kinase gene, mediates osmotic stress and ABA signal responses. J. Plant Physiol. 2012, 169, 1501–1510. [CrossRef] 71. Li, Y.; Zhang, L.; Lu, W.; Wang, X.; Wu, C.A.; Guo, X. Overexpression of cotton GhMKK4 enhances disease susceptibility and affects abscisic acid, gibberellin and hydrogen peroxide signalling in transgenic Nicotiana benthamiana. Mol. Plant Pathol. 2014, 15, 94–108. [CrossRef][PubMed] 72. Greco, M.; Chiappetta, A.; Bruno, L.; Bitonti, M.B. In Posidonia oceanica cadmium induces changes in DNA methylation and chromatin patterning. J. Exp. Bot. 2012, 63, 695–709. [CrossRef][PubMed] 73. Hõrak, H. Defense, fast and slow: Activation of different MAPK pathways in response to wounding. Plant Cell 2020, 32, 1788–1789. [CrossRef][PubMed] 74. Liu, J.; Wang, X.; Yang, L.; Nan, W.; Ruan, M.; Bi, Y. Involvement of active MKK9-MAPK3/MAPK6 in increasing respiration in salt-treated Arabidopsis callus. Protoplasma 2020, 257, 965–977. [CrossRef] 75. Chen, J.; Zhang, Q.; Wang, Q.; Feng, M.; Li, Y.; Meng, Y.; Zhang, Y.; Liu, G.; Ma, Z.; Wu, H.; et al. RhMKK9, a rose MAP KINASE KINASE gene, is involved in rehydration-triggered ethylene production in rose gynoecia. BMC Plant Biol. 2017, 17, 1–11. [CrossRef][PubMed] 76. Shinozaki, K.; Mizoguchi, T.; Irie, K.; Hirayama, T.; Hayashida, N.; Yamaguchi-Shinozaki, K.; Matsumoto, K. A gene encoding a mitogen-activated protein kinase kinase kinase is induced simultaneously with genes for a mitogen-activated protein kinase and an S6 ribosomal protein kinase by touch, cold, and water stress in Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 1996, 93, 765–769. [CrossRef] 77. Ichimura, K.; Mizoguchi, T.; Yoshida, R.; Yuasa, T.; Shinozaki, K. Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J. 2000, 24, 655–665. [CrossRef] 78. Jin, S.L.; Kyung, W.H.; Bhargava, A.; Ellis, B.E. Comprehensive analysis of protein-protein interactions between Arabidopsis MAPKs and MAPK kinases helps deﬁne potential MAPK signalling modules. Plant Signal. Behav. 2008, 3, 1037–1041. [CrossRef] 79. Singh, R.; Jwa, N.S. The rice MAPKK-MAPK interactome: The biological signiﬁcance of MAPK components in hormone signal transduction. Plant Cell Rep. 2013, 32, 923–931. [CrossRef] 80. Song, Q.; Li, D.; Dai, Y.; Liu, S.; Huang, L.; Hong, Y.; Zhang, H.; Song, F. Characterization, expression patterns and functional analysis of the MAPK and MAPKK genes in watermelon (Citrullus lanatus). BMC Plant Biol. 2015, 15, 298. [CrossRef] 81. Shi, J.; An, H.L.; Zhang, L.; Gao, Z.; Guo, X.Q. GhMPK7, a novel multiple stress-responsive cotton group C MAPK gene, has a role in broad spectrum disease resistance and plant development. Plant Mol. Biol. 2010, 74, 1–17. [CrossRef][PubMed] 82. Cheng, F.; Liu, S.; Wu, J.; Fang, L.; Sun, S.; Liu, B.; Li, P.; Hua, W.; Wang, X. BRAD, the genetics and genomics database for Brassica plants. BMC Plant Biol. 2011, 11, 136. [CrossRef][PubMed] 83. Altschul, S.F.; Madden, T.L.; Schäffer, A.A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D.J. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res. 1997, 25, 3389–3402. [CrossRef][PubMed] 84. Mistry, J.; Finn, R.D.; Eddy, S.R.; Bateman, A.; Punta, M. Challenges in homology search: HMMER3 and convergent evolution of coiled-coil regions. Nucleic Acids Res. 2013, 41, e121. [CrossRef][PubMed] 85. Brum, I.J.B.; Martins-de-souza, D.; Smolka, M.B.; Novello, J.C.; Galembeck, E.; De Bioquímica, D.; De Biologia, I.; Paulo, S. Web Based Theoretical Protein pI, MW and 2DE Map. J. Comput. Sci. Syst. Biol. 2009, 02, 93–96. [CrossRef] 86. Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular evolutionary genetics analysis across computing platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [CrossRef][PubMed] 87. Chen, C.; Chen, H.; Zhang, Y.; Thomas, H.R.; Frank, M.H.; He, Y.; Xia, R. TBtools: An Integrative Toolkit Developed for Interactive Analyses of Big Biological Data. Mol. Plant 2020, 13, 1194–1202. [CrossRef] 88. Bailey, T.L.; Boden, M.; Buske, F.A.; Frith, M.; Grant, C.E.; Clementi, L.; Ren, J.; Li, W.W.; Noble, W.S. MEME Suite: Tools for motif discovery and searching. Nucleic Acids Res. 2009, 37, 202–208. [CrossRef]